Kigamicin生物合成基因簇中orf48和orf49的功能研究

发布时间：2018-05-18 06:18

  本文选题：Kigamicin + 生物合成基因簇　； 参考：《福建师范大学》2016年硕士论文

【摘要】：Kigamicin是由Amycolatopsis sp. ML630-mF1发酵产生的一类多环氧杂蒽酮类抗生素,具有抗细菌、抗真菌和特异性抗肿瘤的活性。其特异性抗肿瘤活性表现为可特异性抑制营养饥饿状态下肿瘤细胞,而对正常状态下的细胞无抑制作用,因此可作为特异性抗癌药物的潜在开发来源。鉴于Kigamicin独特的化学结构和生物活性,人们对其结构与功能的关系产生了浓厚的研究兴趣。本研究在Kigamicin生物合成基因簇成功克隆的基础上,通过分子生物学手段对其相关基因功能进行研究,可使我们进一步了解多氧环杂蒽酮类抗生素生物合成机制,为解释其独特的生物活性提供理论依据。首先,通过生物信息学分析推测Kigamicin生物合成基因簇中orf48和orf49的基因功能,orf48与Xantholipin和Lysolipin生物合成基因xanK和llpK同源性较高,初步推测orf48可能编码3Fe-S Ferredoxin,参与Kigamicin生物合成途径中的电子传递和氧化还原反应；orf49与Xantholipin生物合成基因xan02功能类似,有可能负责Kigamicin生物合成中亚甲基双氧桥的形成；但是也可能与Lysolipin生物合成基因llpOIV相似,参与催化Kigamicin中氧杂葸酮环形成前的环氧化反应。然后,通过PCR-targeting技术分别构建缺失orf48质粒和缺失orf49质粒,即分别以壮观霉素抗性基因替换质粒pLL59上的orf48和orf49,并通过抗性筛选转化子,经PCR验证后,得到突变质粒pLL214和pLL215。而后通过接合转移将突变质粒分别转入天蓝色链霉菌ML154,抗性筛选得到突变株Kiga21468和突变株Kiga21568.通过HPLC-MS检测突变株Kiga21468、Kiga21568以及野生型ML154发酵产物,结果比对,发现突变株Kiga21468和突变株Kiga21568产生了相同的物质组分,并且与Kigamicin具有相同UV特征吸收峰。由此推测,突变株Kiga21468和Kiga21568可能产生Kigamicin结构类似物。对突变株发酵积累代谢产物[M+H]/Z=497, [M+H]/Z=538, [M+H]/Z=527进行分离纯化和结构鉴定,化合物[M+H]/Z=538的分子式为C28H27NO10,其化学结构与Kigamicin结构相比缺少亚甲基双氧桥结构,故而推测Orf48和Orf49参与了Kigamicin亚甲基双氧桥结构的生物合成。
[Abstract]:Kigamicin is made by Amycolatopsis sp. A class of polycyclic oxyanthrone antibiotics produced by ML630-mF1 fermentation, with antibacterial, antifungal and specific antitumor activities. Its specific antitumor activity is that it can specifically inhibit tumor cells in the condition of nutritional starvation, but has no inhibitory effect on the cells in normal state, so it can be used as a potential source for the development of specific anticancer drugs. In view of the unique chemical structure and biological activity of Kigamicin, the relationship between its structure and function has attracted much attention. On the basis of the successful cloning of Kigamicin biosynthesis gene cluster, the function of its related genes was studied by molecular biological methods, so that we can further understand the biosynthesis mechanism of polyoxocyclic anthrone antibiotics. It provides theoretical basis for explaining its unique biological activity. Firstly, the gene function of orf48 and orf49 in Kigamicin biosynthesis gene cluster is conjectured by bioinformatics analysis. The gene function of orf48 and orf49 is highly homologous to that of Xantholipin and Lysolipin biosynthesis gene xanK and llpK. It is inferred that orf48 may encode 3Fe-S Ferredoxin, which is involved in the electron transport and redox reaction in the Kigamicin biosynthesis pathway, and may be responsible for the formation of methylene dioxide-bridge in Kigamicin biosynthesis, which is similar to that of Xantholipin biosynthesis gene xan02. But it may also be similar to the Lysolipin biosynthesis gene llpOIV, which is involved in the epoxidation reaction before the formation of oxa-anthracanone ring in Kigamicin. Then, the deletion orf48 plasmid and the deletion orf49 plasmid were constructed by PCR-targeting technique, that is, the orf48 and orf49 on the plasmid pLL59 were replaced with the spectinomycin resistance gene, and the mutant plasmids pLL214 and pLL215were obtained after PCR verification by screening the transformants. Then the mutant plasmids were transferred into Streptomyces cerevisiae ML154 by conjugation transfer. The mutant Kiga21468 and the mutant Kiga21568 were obtained by resistance screening. The fermentation products of the mutant Kiga21468 and wild-type ML154 were detected by HPLC-MS. The results showed that the mutant Kiga21468 and the mutant Kiga21568 produced the same substance components and had the same UV characteristic absorption peak as Kigamicin. It is inferred that Kiga21468 and Kiga21568 may produce Kigamicin structural analogues. The metabolites of [M H] / r ZN 497, [M H] / R ZN 538, [M H] / R ZN 527 were isolated, purified and identified. The molecular formula of compound [M H] / R ZN 538 is C28H27NO10, and its chemical structure lacks methylene dioxide-bridge structure compared with the structure of Kigamicin. Therefore, it is speculated that Orf48 and Orf49 are involved in the biosynthesis of Kigamicin methylene dioxide-bridge structure.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ927;Q78

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